Tumor necrosis factor-alpha (TNF-.alpha.) is a 17-kd polypeptide released primarily by macrophages. Generally, TNF-.alpha. is not present in measurable quantity in sera from healthy individuals; but appears rapidly in response to immunostimulators (Beutler and Cerami, Adv. Immunol. 42:213-232, 1988). At physiological concentrations, TNF-.alpha. limits the growth and spread of invasive pathogens. However, excessive or uncontrolled production of this cytokine contributes to the pathogenesis of number of disease conditions.
TNF-.alpha., acting alone and/or in concert with other mediators, evokes a potentially fatal syndrome of irreversible cardiovascular collapse (shock) and critical organ failure (Beutler, Science 229:869-871, 1985; Tracy et al., Nature 330:662-664, 1987; Waag et al., Lancet 1:355-357, 1987). Additionally, TNF-.alpha. acting in concert or in synergy with the interleukins IL-1 and IL-6 contributes to the development of wasting syndrome (cachexia) (Grunfeld, et al., Am. J. Clin. Nutr. 55:455-460, 1992; Grunfeld and Feingold, N. Engl. J. Med. 327:329-337, 1992).
For example, the experimental administration of supernatants from endotoxin-stimulated macrophages produced severe weight loss in rodents (Cerami et al., Immunol. Lett. 11:173-177, 1985). Moreover, nude mice implanted with genetically engineered tumor cells that secreted either TNF-.alpha. (Rouzer and Cerami, Mol. Biochem. Parasitol. 2:31-38, 1980) or IL-6 (Black et al., Endocrinology 128:2657-2659, 1991) became progressively anorectic and wasted. Administering TNF-.alpha., IL-1, and IL-6 increases plasma triglycerides in rodents by boosting hepatic lipogenesis and very-low-density lipoprotein production leading to futile cycling of fatty acid/triglyceride and eventually wasting (Feingold and Grunfeld, J. Clin. Invest. 80:184-190, 1987; Grunfeld et al., Cancer Res. 50:4233-4238, 1990; Feingold, et al., Arterioscler. Thromb. Vasc. Biol. 11:495-500, 1991).
Cytokines also boost the levels of key catabolic hormones; alter glucose and amino acid metabolism; and have profound effect on food intake. In experimental animals, TNF-.alpha. decreases gastric motility and consequently leads to retention of food (Patten et al., J. Clin. Invest. 80:1587-1596, 1987), and IL-1 induces continuous anorexia by indirectly affecting the hypothalamic appetite center (Hellerstein et al., J. Clin. Invest. 84:228-235, 1989). In humans, wasting syndrome is often associated with cancer and a variety of infectious diseases including, but not limited to tuberculosis and AIDS.
In addition to progressive weight loss, many patients experience anorexia (reduced appetite), nausea, muscle weakness, and anemia (Lawson et al., Lancet 2:1-5, 1982; Grunfeld and Feingold, New Engl. J. Med. 237:329-337, 1992). Although cachexia may involve anorexia, usually the degree of lean body mass lost in cachexia associated with cancer and infectious disease cannot be explained by reduced caloric intake (Spiegelman and Hotamisligil, Cell 73:625-627, 1993).
Cachexia is considered as a detrimental end point because, apart from directly effecting patient survival, the progressive weight loss and anemia usually restrict the ability of cachectic patients to tolerate aggressive therapy (Dewy et al., Am. J. Med. 69:491-497, 1980).
The prevalence of cachexia makes this syndrome a significant medical problem. Taken together, these results provide a mechanistic basis for considering the use of melanin, an agent that interferes with the synthesis/release of IL-1, IL-6 and TNF-.alpha., for managing wasting in patients.
TNF-.alpha. can also induce adult respiratory distress syndrome (ARDS), a severe consequence of gram-negative sepsis in humans (Shaby et al., In: Pathophysiology of Endotoxin, J. B. Hinshaw, ed. Amsterdam: Elsevier, pp. 105-128, 1985). TNF-.alpha. concentrations in excess of 12,000 pg/ml were detected in pulmonary aspirates from ARDS patients (Millar et al., Lancet 2:712-714, 1989). This cytokine is also known to increase the adherence of polymorphonuclear leukocytes to endothelial cells (Gamble et al., Proc. Natl. Acad. Sci., U.S.A. 82:8667-8671, 1985). Increased adherence of activated granulocytes in the microvasculature of the lungs and upper respiratory tract is one of the major causes of pulmonary vascular injury in ARDS. Of note, expression of intracellular adhesion molecule (ICAM), and endothelial leukocyte adhesion molecule (ELAM) on endothelial cells is either induced or enhanced by cytokines such as TNF-.alpha. or IL-1 (Munre et al., Am. J. Pathol. 135:121-132, 1989), a phenomenon which results in the augmentation of cell binding.
TNF-.alpha., IL-1 and IL-6 also play a major role in the pathology of rheumatoid arthritis (Saklatvala., Nature 322:547-549, 1986; Miossec. Clin. Rheumatol. 5:305-308, 1987; Lupia et al. Eur. J. Immunol., 26: 1690-1694, 1996). Synovial fluids from patients with rheumatoid arthritis contain TNF-.alpha. (Saxne et al., Arthritis Rheumatism 31:1041-1132, 1989) and IL-6 (Guerne et al., J. Clin. Invest. 83:585-592, 1989). Current evidence suggests that immune complexes may stimulate monocytes to secrete TNF-.alpha. (Visser et al., Am. J. Pathol. 134:1-6, 1989) and IL-1 (Chantry et al., Eur. J. Immunol. 19:189-192, 1989). TNF-.alpha. and IL-1 in turn stimulates production of proteases and prostaglandins by synoviocytes and bone resorption by osteoclasts (Miossec, Clin. Rheumatol. 5:305-308, 1987; Dayer et al., J. Exp. Med. 162:2163-2168, 1985; Saklatvala, Nature 322:547-549, 1986). Moreover, the presence of TNF-.alpha. and IL-1 in rheumatoid joints may act together to perpetuate synovitis by stimulating IL-6 synthesis which, if found in close proximity to plasma cells, may lead to autoantibody production. IL-6 is spontaneously produced by synoviocytes and high levels of IL-6 are present in synovial fluids from patients with inflammatory arthropathies (Guerne et al., J. Clin. Invest. 83:585-592, 1989).
Cerebral malaria is a lethal hyperacute neurological syndrome and prognosis of some malaria patients which has been associated with threshold levels of serum TNF-.alpha. (Grau et al., Science 237:1210-1212, 1987; Clark et al. Am. J. Pathol., 129:192-199, 1987; Grau et al., New Engl. J. Med 320:1586-1591, 1989; Kwiatkowski et al., Lancet 336:1201-1204, 1990; McGuire et al. Nature 371-510, 1994). Similarly, in Graft versus Host Reactions, increases in TNF-.alpha. concentration have been associated with major complications (Holler et al., Blood 75:1011-1016, 1990).
TNF-.alpha. alone, or in synergy with either IL-1 or IL-6, enhances replication of HIV-1 in latently infected T cells and monocytes (Folks et al., Science 238:800-802, 1987; Folk et al., Proc. Natl. Acad. Sci. U.S.A. 86:2365-2368, 1989; Poli et al., J. Exp. Med. 172:151-158, 1990; Poli et al., Proc. Natl. Acad. Sci. U.S.A. 91:108-112, 1994). TNF-.alpha. is a strong inducer of NF-.kappa..beta., a transcriptional factor used by HIV (Nobel and Baltimore, N. Engl. J. Med. 234:308-317, 1987; Duh et al., Proc. Natl. Acad. Sci. U.S.A. 86:5974-5978, 1989). Moreover, synthesis of TNF-.alpha., IL-1, and IL-6 are upregulated as a consequence of HIV infection (Folks et al., Science 238:800-802, 1987; Nakajima et al., J. Inmunol. 142:531-536, 1989). Serum and cerebrospinal fluid of patients with AIDS contain increased levels of TNF-.alpha., IL-1, and IL-6 (Lahdevirta et al., Am. J. Med. 85:289-291, 1988; Emille et al., J. Clin. Invest. 86:148-159, 1990; Breen et al., J. Immunol. 144:480-484, 1990).
The apoptotic neuronal loss occurring in HIV-1 encephalitis is associated with TNF-.alpha. (DeSimone et al., Immunol. Today 17:256-258, 1996). In addition, TNF-.alpha. has been implicated in AIDS associated cachexia (Wright et al., J. Immunol. 141:99-104, 1988). Therefore, the downregulation of abnormal cytokine production by monocytes, and particularly the down-regulation of TNF-.alpha. is expected to retard the progression of HIV infection and provide supportive care for cachexic patients.
IL-6 is also an autocrine growth factor for cells derived from Kaposi sarcoma (KS) lesions of patients with AIDS (Miles et al., Proc. Natl. Acad. Sci. 87:4068-4072, 1990). KS, a multifocal vascular lesion, is also seen in other immunosuppressed states such as in patients receiving renal or cardiac transplants (Gang and Jones, Clin. Exp. Dermatol. 3:135-146, 1978; Greenfield et al., J. Rheumatol. 13:637-640, 1986). AIDS-KS-derived cell lines contain and secrete substantial amounts of IL-6 and AIDS-KS growth-enhancing effects of tat protein are mediated by increased IL-6 production. Indeed, addition of IL-6 antisense oligodeoxynucleotides to these cells resulted in decreased IL-6 production as well as marked inhibition of their growth (Miles et al., Proc. Natl. Acad. Sci. 87:4068-4072, 1990).
AIDS-KS derived cells produce other cytokines including IL-1 (Marx, Science 248:442-443, 1990) Addition of anti-IL-1 antibody to KS cell lines also resulted in decreased cellular proliferation. The increased levels of serum IL-6 and polyclonal B cell activation may be associated with increased frequency of B cell malignancies seen in AIDS patients (Akira and Kishimoto, Immunol. Rev. 127:26-50, 1992).
Like KS, the existence of an IL-6-IL-6-receptor autocrine loop has been implicated in the pathogenesis of multiple myeloma (Kawano et al., Nature 332:83-85, 1988). Elevated levels of IL-6 have been observed in other pathological conditions such as mesangial proliferative glomerulonephritis (Horii et al., J. Immunol. 143:3949-3955, 1989), and psoriasis (Grossman et al., Proc. Natl. Acad. Sci. 86:6367-6371, 1989).
A wide variety agents have been used to combat inflammation and life-threatening aspects of cytokines. Anti-TNF-.alpha. antibody, the TNF-.alpha. receptor, anti-IL-6, and IL-1 receptor antagonist (IL-1Ra) therapy were shown to reduce death after acute systemic toxicity (e.g., septic shock) in experimental animals (Beutler et al., Science 229:869-871, 1985; Tracy et al., Nature 330:662-664, 1987; Ohlsson et al., Nature 348:550-552, 1990; Starnes et al., J. Immunol. 145:4185-4191, 1990; Ashenazi, et al., Proc. Natl. Acad. Sci. U.S.A. 88:10535-10539, 1991; Lesslaner et al., Eur. J. Immunol. 21:2883-2886, 1991). However, the response to these cytokine blockers depended on the prophylactic administration of the agent, or the site of infection (Bagby et al., J. Infect. Dis. 163:83-88, 1991). Moreover, in a number of studies, anti-cytokine antibodies only partially protected the animals (Feingold et al., J. Clin. Invest. 83:1116-1121, 1989).
Data from several studies indicated that blockade of cytokines by infusion of either anti-TNF-.alpha. (Elliott et al., Arthritis Rheum. 36:1681-1690, 1993; Elliott et al., Lancet 344:1105-1110, 1994), or anti-IL-6 (Wendling et al., J. Rheumatol. 20:259-262) monoclonal antibody, as well as soluble TNF-.alpha. receptors (Moreland et al., Arthritis Rheum. 37:S295, 1994), or soluble IL-1 receptor (Drevlow et al., Arthritis Rheum. 37:S339, 1994) is effective in the treatment of rheumatoid arthritis. However, use of soluble cytokine receptors or antibodies to a single factor is constrained by the presence of multiple cytokines that participate in the manifestation of inflammatory conditions. Moreover, the large-scale treatment with anticytokine antibody may lead to production of anti-idiotypic antibodies.
Agents such as dexamethasone (Luce et al., Am. Rev. Respir. Dis. 138:62-68, 1988), pentoxifylline (Netea et al., J. Infect. Dis. 171:393-399, 1995), thalidomide (Klausner et al., J. Acquir. Immun. Defic. Syndr. Hum. Retrovirol. 11:247-257, 1996), suramin (Strassman et al., J. Clin. Invest 92:2152-2159, 1993), or .alpha.-melanocyte-stimulating hormone (.alpha.-MSH) (Chio et al., J. Clin. Invest. 97:2038-2044, 1996) have also been used for limiting synthesis of proinflammatory cytokines. With the exception of .alpha.-MSH, these agents have limited clinical utility because they are either ineffective when given after challenge (dexamethasone and pentoxifylline), do not target multiple cytokines, or have multiple side effects.
Thalidomide and pentoxifylline inhibit production of TNF-.alpha. but not IL-1.beta. or IL-6 (Sampaio et al., J. Exp. Med. 173:699-703, 1991). Because multiple cytokines contribute to the pathogenesis of inflammatory disorders, inhibition of a single cytokine may not reverse or prevent the progression of disease. Thalidomide is teratogenic and has been used in the past as a sedative and antiemetic, and suramin has considerable toxicity (Stein, Cancer Res. 53:2239-2248, 1993).
Corticosteroids, the mainstay anti-inflammatory agents, manifest adverse effects such as susceptibility to infection, suppression of the hypothalamic-pituitary-adrenal axis, and Cushingoid features. Use of cyclosporine A may result in hypertension and nephrotoxicity.
Melanin, inter alia, is a free radical scavenger that acts as a bacterial virulence factor by protecting the organism from some host defense mechanisms (Wang and Casadevail, Infect. Immun. 62:3004, 1994). Additional studies have shown that melanin expression by bacteria may be a virulence factor that helps bacterial pathogens avoid the afferent phase of T cell-mediated immune responses in the host (Huffnagle et al., J. Immunol. 155:3507-3516, 1995).